LOCUS       sequence-371945-        4748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ratiometric reporter for cAMP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4748)
  AUTHORS   Kawata S, Mukai Y, Nishimura Y, Takahashi T, Saitoh N
  TITLE     Green fluorescent cAMP indicator of high speed and specificity 
            suitable for neuronal live-cell imaging.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Jul 12;119(28):e2122618119. doi: 
            10.1073/pnas.2122618119. Epub 2022 Jul 6.
  PUBMED    35867738
REFERENCE   2  (bases 1 to 4748)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2022-07-12"; volume: "119(28):e2122618119. 
            doi"; pages: " 10.1073/pnas.2122618119. Epub 2022 Jul 6"
COMMENT     Sequence Label: pUC57-rm_gCarvi-T2A
FEATURES             Location/Qualifiers
     source          1..4748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(471..524)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     658..677
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1044..1065)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1692..1757)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      1757..1766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1788..2495)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2063..2082)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2332..2350
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2527..2543)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2527..2543)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2540..2562)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2551..2567
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2575..2581,2582..2599,2600..2605))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2620..2641
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2758..2775)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2929..3517)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3009..3028)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3688..4479,4480..4548))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4311..4330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4549..4653)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4721..4739
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tcccaatgtc gacggtatcg ataagcttga tatcgaattc agggccaggg
      481 ttttcctcca cgtcgccgca ggtcagcagg ctgcctctgc cctcagatcc ctcgacaagc
      541 ttactagtgt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      601 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      661 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggatgtagcc ttcgggcatg
      721 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      781 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      841 atgaacttca gggtcagctt gccgtaggtg gcatcgccct caccctcgcc ggacacgctg
      901 aacttgtggc cgtttacgtc gccgtccagc tcgacctgga tgggcaccac cccggtgaac
      961 agctcctcgc ccttgctcac catgctccct ccggtaccgc ccttgtacag ctcgtccatg
     1021 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     1081 ttggggtctt tcgaaagaat ggactgcacg ctcaggtagt ggttgtcggg cagcagcacg
     1141 gggccgtcgc cgatgggggt gttctgctgg tagtggtagg cgagctgcac gccgccgtcc
     1201 tcgatgttgt ggcggatctt gaagttcgcc ttgatgccgt tcttctgctt gtcggccttg
     1261 atatagacgt tctcgaggta caggttcccc accttctcag aggtaacctg aagccttctc
     1321 gccatctggg ctgagaggcg catgagtatg tcgggattca cctgtatgag ttgccggaat
     1381 ttcttgtatg agatttcggc cacctcacag gctgtcttag ccctcaccca agcggatctc
     1441 tcctgtcctt cctcgaacag gcccagttct ccgataaagt caccctggtt cagatagctc
     1501 aggatcattt cctttccttc ttcatcctta atcagcacgg cgactgaacc cttcactata
     1561 taatacaggg tttcagcctt ctcgccctga tggatcaggg tgctcttaga tgggtacttg
     1621 tgaatgtgac agtgtgacag aaaccactcc aatgttgggt cggtctgtgg tttgcccagc
     1681 accatggatc ccttgtcatc gtcatccttg taatcgatgt catgatcttt ataatcaccg
     1741 tcatggtctt tgtagtccat ggtggcacta gtagatccga gtccggactt gtacagctcg
     1801 tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt actgttccac gatggtgtag
     1861 tcctcgttgt gggaggtgat gtccaacttg atgttgacgt tgtaggcgcc gggcagctgc
     1921 acgggcttct tggccttgta ggtggtcttg acctcagcgt cgtagtggcc gccgtccttc
     1981 agcttcagcc tctgcttgat ctcgcccttc agggcgccgt cctcggggta catccgctcg
     2041 gaggaggcct cccagcccat ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg
     2101 gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct gcagggagga gtcctgggtc
     2161 acggtcacca cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg
     2221 aaggacagct tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc cttggagccg
     2281 tacatgaact gaggggacag gatgtcccag gcgaagggca gggggccacc cttggtcacc
     2341 ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg
     2401 aactcgtggc cgttcacgga gccctccatg tgcaccttga agcgcatgaa ctccttgatg
     2461 atggccatgt tatcctcctc gcccttgctc accatggtag atccactagt tctagtagat
     2521 ctcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2581 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2641 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2701 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2761 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2821 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2881 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2941 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3001 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3061 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3121 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3181 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3241 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3301 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3361 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3421 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3481 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3541 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3601 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3661 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3721 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3781 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3841 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3901 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3961 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4021 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4081 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4141 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4201 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4261 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4321 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4381 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4441 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4501 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4561 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4621 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4681 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     4741 ctttcgtc
//
